Effects of Nitrogen Deposition and Precipitation Patterns on Nitrogen Allocation of Mongolian Pine (Pinus sylvestris var. mongolica) on Sandy Land Using ¹⁵N Isotope

Abstract

In the context of global climate change, atmospheric nitrogen deposition is increasing, and precipitation patterns are becoming more variable. This study examines the impact of these changes on nitrogen (N) allocation mechanisms in semi-arid region tree species using one-year-old Mongolian pine (Pinus sylvestris var. mongolica) seedlings. The seedlings were planted in soil collected from the Daqinggou Sandy Ecological Experiment Station (42°54′ N, 122°25′ E). Three moisture treatments were applied (WC (normal moisture, approximately 65% ± 2.5% of the field capacity), WI (30% increased moisture), and WD (30% decreased moisture)), as well as three nitrogen treatments (NC (no nitrogen), NL (5 g·m⁻²·y⁻¹ nitrogen), and NH (10 g·m⁻²·y⁻¹ nitrogen)). The seedlings were sprayed with a ¹⁵N-labeled CH₄N₂O solution (46% N, ¹⁵N abundance 10.14%) in a pot trial, with samples taken in August and October to measure N content and ¹⁵N abundance in the seedling organs and the soil. Parameters such as N_dff (%) (the percentage of nitrogen derived from fertilizer), nitrogen content of organs, ¹⁵N absorption in organs, and ¹⁵N distribution ratio were calculated. The results showed that ¹⁵N allocation in seedlings followed the trend leaves > stems > roots. Under moisture treatments, ¹⁵N allocation ratios in leaves, stems, and roots were 63.63–71.42%, 14.89–24.14%, and 12.23–14.88% under low nitrogen, and 62.63–77.83%, 13.35–22.90%, and 7.31–19.18% under high nitrogen. Significant correlations were found in ¹⁵N abundance among the seedling organs, with coefficients ranging from 0.97 to 1.00. The main effects of moisture and nitrogen, as well as their interaction, significantly impacted ¹⁵N abundance in the seedling organs. Changes in moisture levels affected the nitrogen absorption capacity of Mongolian pine. Increased moisture significantly enhanced ¹⁵N absorption in all organs, leading to 62.63–71.42% of ¹⁵N being allocated to the leaves, maintaining an appropriate proportion with the roots and stems. Nitrogen deposition altered the nitrogen allocation strategy among different organs of Mongolian pine. Under conditions of reduced moisture and low nitrogen, a greater proportion of nitrogen was captured by the roots and stems, with an allocation increase of approximately 4.98–5.77% compared to the control group, thereby mitigating the adverse effects of water deficiency. In conditions of reduced moisture and high nitrogen, the leaves, being active organs, accumulated more limiting elements, with an increase in nitrogen allocation of 2.03–8.07% compared to the control group. To achieve an optimal allocation strategy, moderate nitrogen deposition combined with increased moisture enhanced nitrogen uptake in Mongolian pine seedlings. This study provides scientific evidence for ecological restoration, wind erosion control, and agricultural and forestry management in semi-arid regions under the context of global climate change.

1. Introduction

In recent years, economic and social development, along with human activities, have exacerbated global climate change. Among these changes, nitrogen (N) deposition and variable precipitation patterns have garnered widespread attention. Existing studies indicate that the frequency of short-term extreme increases in global summer rainfall events has risen [1,2], often followed by consecutive droughts [3]. These complex precipitation patterns alter the allocation strategies of elements within plants [4], threatening ecosystem functions and the stability of plant communities [5,6]. Anthropogenic nitrogen emissions, such as those from the industrialization of agriculture and livestock, are contributing to increasing atmospheric nitrogen deposition [7]. Current global estimates of total nitrogen deposition range between 125 and 132 TgN yr⁻¹ [8], posing a severe air pollution problem [9]. Research has shown that atmospheric nitrogen deposition affects the growth and development of plants and animals, impairing ecosystem functions [10,11]. Continuous increases in nitrogen deposition impact biochemical processes, such as carbon and nitrogen cycling in forest ecosystems [12,13], influence nitrogen cycling in marine ecosystems, and inhibit marine productivity [14]. It is the third largest cause of global biodiversity loss [15] and a significant factor threatening the health of natural ecosystems and the biosphere [16].

Climate change has intensified the process of desertification, particularly in the border area between western Liaoning Province and the Horqin Sandy Land in eastern Inner Mongolia. This region is experiencing increasing desertification and has sensitive and fragile ecosystems [17]. The frequency of mild and moderate droughts in this area is 48.16% and 38.41%, respectively [18]. Research indicates that N, a crucial limiting element for plant growth [19], exhibits storage and distribution strategies within plants that are more sensitive to external nutrients and climate change in semi-arid environments [20,21], employing unique mechanisms [22]. Plants growing in semi-arid regions can increase nitrogen uptake to mitigate some of the water deficit [23], thereby enhancing their adaptability to water scarcity [24]. This has led us to focus our research on the response mechanisms of plants in semi-arid regions under the influences of nitrogen deposition and changes in precipitation patterns.

Mongolian pine (Pinus sylvestris var. mongolica) is an evergreen tree species known for its resistance to cold, drought, and poor soil conditions. It is currently being planted in the border area between western Liaoning Province and the Horqin Sandy Land in eastern Inner Mongolia as an ecological species to improve soil quality [25] and mitigate soil desertification [26,27]. Compared to other tree species, Mongolian pine is more sensitive to changes in precipitation and other climate variables [28,29]. This region is experiencing significant increases in nitrogen deposition [2] and overall trends of rising annual average temperatures and decreasing precipitation [30]. In individual years, there may also be brief periods of heavy rainfall or increased precipitation due to specific climatic conditions [31]. This complex precipitation pattern, combined with atmospheric nitrogen deposition, creates composite effects that warrant our attention. However, most current research predominantly focuses on drought stress in arid and semi-arid regions [32], with few studies examining the effects of increased water input. Existing studies have shown that nutrient changes due to nitrogen deposition are increasing plant nitrogen uptake while altering the distribution strategies of nitrogen and other elements [33]. Additionally, moisture availability affects the usability of nitrogen within plants [34]. Under the compounded long-term effects of atmospheric nitrogen deposition and changes in precipitation patterns, it is crucial to monitor the responses of Mongolian pine to external climatic changes such as nitrogen and moisture when using it for ecological purposes [35]. For this purpose, we employed stable-isotope tracing technology, which is widely used to reveal the dynamics of nitrogen distribution within plants [36,37], to assess the intrinsic effects of increased exogenous nitrogen concentrations from nitrogen deposition and fertilizers on plant nutrient utilization [38].

Regarding the border area between western Liaoning Province and southern Horqin Sandy Land, there are few studies on the combined effects of intensified nitrogen deposition and variable precipitation patterns on the nitrogen distribution of ecological forest trees like Mongolian pine; most focus on investigating changes in ecological stoichiometric characteristics of ecological forest trees resulting from moisture or nitrogen deposition [39]. This study employs stable-isotope tracing technology, using foliar application of ¹⁵N-labeled urea to simulate atmospheric nitrogen deposition and varying soil moisture to represent changes in precipitation patterns. The focus is on exploring the nitrogen distribution strategies of Mongolian pine under the combined influence of changing precipitation patterns and atmospheric nitrogen deposition, with some degree of novelty. The aim is to provide theoretical references for ecological restoration and desertification control in the context of global climate change.

2. Materials and Methods

2.1. Experimental Materials

This study used one-year-old Mongolian pine (Pinus sylvestris var. mongolica) seedlings as experimental materials. The soil was collected from the Daqinggou Sandy Land Ecological Experiment Station (42°54′ N, 122°25′ E) of the Institute of Applied Ecology, Chinese Academy of Sciences. This station is located at the border between western Liaoning Province and the southern part of the Horqin Sandy Land in Inner Mongolia. The area features a continental temperate semi-humid-to-semi-arid transitional climate. The mean annual temperature (MAT) is 6.4 °C, and the mean annual precipitation (MAP) is 441 mm [40]. The soil at the study site is a nutrient-poor sandy type, consisting of 90.9% sand, 5.0% silt, and 4.1% clay [41]. The soil pH is about 6.6, bulk density (BD) is 1.47 g·cm³, and total nitrogen content is 0.36 g·kg⁻¹ [31].

2.2. Experimental Design

The experiment utilized 30 cm diameter and 30 cm high pots with a sealed bottom and no solution leakage, each filled with 15 kg of soil (approximately up to 28 cm of the pot’s height). Mongolian pine seedlings with uniform growth were transplanted into the pots. The seedlings were grown under natural conditions. On rainy days, they were placed under a rain shelter connected to the natural environment on all sides, ensuring proper ventilation. The shelter was only shaded on the upper part, which excluded external factors such as rainfall and temperature.

Based on historical soil moisture data from the Daqinggou Sandy Land Ecological Experiment Station [31,42], we established three water treatment levels based on the soil’s field capacity: WC (normal moisture, approximately 65% ± 2.5% of the field capacity), WI (30% increased moisture, approximately 85% ± 2.5% of the field capacity), and WD (30% decreased moisture, approximately 45% ± 2.5% of the field capacity). The moisture control trial commenced on May 1 and concluded on October 30 of the same year. Soil moisture content was measured using a soil moisture meter (TDR300, Spectrum Technologies, Inc., Plainfield, IL, USA) and controlled by the weighing method. Before we started the water control test, we watered it thoroughly. When the soil moisture content reached the level specified by the experimental design, the total weight of the potting bucket, soil, and seedlings (referred to as Control Weight, CW) was recorded. Due to transpiration, evaporation from the seedlings, and soil evaporation, the soil moisture content decreased over time. Therefore, every three days, we measured the total weight of the potting bucket, soil, and seedlings (referred to as Total Weight, TW) and replenished the water in the potting buckets; the amount of water added was recorded until the end of October. The amount of water replenished (referred to as Water Weight, WW) was determined using the following formula:

$$\mathrm{W}\mathrm{W}=\mathrm{C}\mathrm{W}-\mathrm{T}\mathrm{W}$$

Referring to ambient N deposition fluxes in northern China [43] and monitoring data from the Nationwide Nitrogen Deposition Monitoring Network (NNDMN) from 2010 to 2014 in eastern Inner Mongolia and northeastern China [44], combined with data from the study site [45] and considering the ecological effects of increased nitrogen deposition in the future, three nitrogen levels were established: NC (control nitrogen treatment, no nitrogen added), NL (5 g·m⁻²·y⁻¹ nitrogen added, equivalent to 50 kg·ha⁻¹·y⁻¹), and NH (10 g·m⁻²·y⁻¹ nitrogen added, equivalent to 100 kg·ha⁻¹·y⁻¹), which are comparable to the treatment levels in existing research settings [40,46,47]. To mimic the process of atmospheric nitrogen deposition, a solution of ¹⁵N-labeled CH₄N₂O (containing 46% N with a ¹⁵N abundance of 10.14%, from Shanghai Research Institute of Chemical Industry Co., Ltd., Shanghai, China) was sprayed evenly on 15 May and 15 June, respectively, during the growing season. The total amount of nitrogen applied was equivalent to the level of the experimental design.

2.3. Measurement and Calculation of Indicators

2.3.1. Sample Collection and Dry Weight Measurement

In the summer (31 August) and autumn (31 October) of the same year, three seedlings from each treatment level were collected. The seedlings were washed with deionized water and divided into roots, stems, and leaves. Surface soil samples were also taken.

The root, stem, and leaf samples were placed in an oven, inactivated at 105 °C for 30 min, and then dried at 70 °C to a constant weight. The dry weights of the root, stem, and leaf samples were then measured and recorded.

2.3.2. N Content and ¹⁵N Abundance Measurement

The dried root, stem, and leaf samples were ground and sieved using a grinder, then stored in zip-lock bags in a dry environment. The surface soil samples were air-dried and stored in zip-lock bags in a dry environment.

The N content and ¹⁵N abundance were measured using a stable-isotope ratio mass spectrometer (Isoprime100, Isoprime Ltd., Cheadle, Greater Manchester, UK).

2.3.3. Calculation

N_dff (%) represents the proportion of nitrogen in each plant organ derived from the applied fertilizer nitrogen, reflecting the absorption capacity of ¹⁵N by each organ [48]. It is calculated according to the formula by Zhao et al. [49], with the natural abundance of ¹⁵N being approximately 0.366%:

$${\mathrm{N}}_{\mathrm{d}\mathrm{f}\mathrm{f}} \left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{a}\mathrm{b}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} {}^{15}\mathrm{N} \mathrm{i}\mathrm{n} \mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n} - \mathrm{a}\mathrm{b}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} {}^{15}\mathrm{N} \mathrm{i}\mathrm{n} \mathrm{n}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}}{\mathrm{a}\mathrm{b}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} {}^{15}\mathrm{N} \mathrm{i}\mathrm{n} \mathrm{f}\mathrm{e}\mathrm{r}\mathrm{t}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{r} - \mathrm{a}\mathrm{b}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{o}\mathrm{f} {}^{15}\mathrm{N}\mathrm{i}\mathrm{n}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}}}\times 100\mathrm{\%}$$

The nitrogen content of organs (mg/plant) represents the amount of nitrogen in each plant organ and is calculated using the following formula [50]:

$$\mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{s} \left(\mathrm{m}\mathrm{g}/\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\right) = \mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\left(\mathrm{g}/\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\right) \times \mathrm{N} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\left(\mathrm{\%}\right) \times 1000$$

¹⁵N absorption in organs (mg/plant) represents the proportion of ¹⁵N in the nitrogen content of each organ absorbed from the fertilizer. The calculation formula, by Zhao et al. [38], is as follows:

$${}^{15}\mathrm{N} \mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{s}\left(\mathrm{m}\mathrm{g}/\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\right)={\mathrm{N}}_{\mathrm{d}\mathrm{f}\mathrm{f}}\left(\mathrm{\%}\right) \times \mathrm{N}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{s}\left(\mathrm{m}\mathrm{g}/\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\right)$$

¹⁵N distribution ratio (%) represents the proportion of ¹⁵N absorbed by each organ in the total ¹⁵N absorbed by the plant. The calculation formula, by Ding et al. [51] and Shi et al. [52], is as follows:

$${}^{15}\mathrm{N} \mathrm{d}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{b}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o} \left(\%\right)={\displaystyle \frac{{}^{15}\mathrm{N} \mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{s}\left(\mathrm{m}\mathrm{g}/\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\right)}{{}^{15}\mathrm{N} \mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{p}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\left(\mathrm{m}\mathrm{g}/\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}\right)}}\times 100\%$$

2.4. Data Processing

Data organization and processing were performed using Microsoft Excel 2016. Graph plotting and image processing were carried out using Origin 2021. Multifactor ANOVA (analysis of variance) was conducted using SPSS 26.0 to analyze the effects of moisture, nitrogen, months, and their interactions on ¹⁵N abundance in the organs and soil of Mongolian pine. Significance analysis was performed using the Waller–Duncan method and One-Way ANOVA, while correlation analysis was conducted using the Pearson index. The experimental data are presented as mean ± standard deviation.

3. Results

3.1. Impact of Nitrogen Deposition and Precipitation Patterns on ¹⁵N Abundance in Mongolian Pine

3.1.1. Changes in ¹⁵N Abundance in Seedlings

As shown in

Table 1. Impact of nitrogen deposition and precipitation patterns on ¹⁵N abundance (%) in different organs of Mongolian pine seedlings on sandy land.

Month	Organs	Treatments
		WCNC	WINC	WDNC	WCNL	WINL	WDNL	WCNH	WINH	WDNH
August	Root	0.03 ± 0.00 A c	0.03 ± 0.00 A c	0.05 ± 0.00 A c	2.89 ± 0.01 B b	4.19 ± 0.03 A b	2.37 ± 0.00 C b	2.78 ± 0.01 C a	5.53 ± 0.11 A a	3.97 ± 0.04 B a
	Stem	0.08 ± 0.01 A c	0.05 ± 0.00 A c	0.07 ± 0.01 A c	4.11 ± 0.02 B b	4.45 ± 0.03 A b	2.93 ± 0.06 C b	4.02 ± 0.06 C a	6.56 ± 0.04 A a	5.08 ± 0.02 B b
	Leaf	0.02 ± 0.00 A c	0.07 ± 0.00 A c	0.02 ± 0.02 A c	3.45 ± 0.01 B b	3.61 ± 0.01 A b	2.46 ± 0.03 C b	3.83 ± 0.04 C a	6.70 ± 0.04 A a	5.04 ± 0.22 B a
October	Root	0.28 ± 0.01 A c	0.21 ± 0.01 A c	0.29 ± 0.01 A c	4.71 ± 0.04 B b	5.49 ± 0.06 A b	3.72 ± 0.00 C b	5.16 ± 0.02 B a	7.93 ± 0.12 A a	3.59 ± 0.18 C a
	Stem	0.20 ± 0.02 A c	0.21 ± 0.00 A c	0.25 ± 0.00 A c	4.88 ± 0.01 B b	5.58 ± 0.03 A b	3.89 ± 0.02 C b	5.82 ± 0.07 B a	7.59 ± 0.06 A a	3.78 ± 0.08 C a
	Leaf	0.03 ± 0.00 A c	0.05 ± 0.00 A c	0.09 ± 0.00 A c	4.30 ± 0.13 B b	5.53 ± 0.01 A b	3.57 ± 0.07 C a	5.20 ± 0.01 B a	8.13 ± 0.01 A a	3.55 ± 0.03 C a

WC (normal moisture), WI (30% increased moisture), and WD (30% decreased moisture). NC (control nitrogen treatment, no nitrogen added), NL (5 g·m⁻²·y⁻¹ nitrogen added), and NH (10 g·m⁻²·y⁻¹ nitrogen added). Different uppercase letters in the same row indicate that the differences between different moisture treatments reached a significant level (p < 0.05) under the same nitrogen treatment. Different lowercase letters in the same row indicate that the differences between the different nitrogen treatments reached a significant level (p < 0.05) under the same moisture treatment.

, in August, under the NC treatment, the three moisture gradient treatments did not cause significant differences in the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings. The ¹⁵N abundance was negligible. Under the NL treatment, compared to the control group, the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings increased or decreased with changes in moisture levels, exhibiting significant differences under different moisture treatments. In the NH treatment, regardless of whether moisture levels increased or decreased, the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings was higher than in the control group, with significant differences observed across the three moisture treatments. Under the WC treatment, the ¹⁵N abundance in the roots and stems was slightly lower under the high nitrogen concentration (NH) treatment compared to the low nitrogen concentration (NL) treatment, whereas it was the opposite in the leaves. Under the increased moisture treatment (WI), the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings increased by 31.98%, 47.42%, and 85.59%, respectively, in the NH treatment compared to the NL treatment, with significant differences. Under the decreased moisture treatment (WD), the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings increased by 67.51%, 73.38%, and 104.88%, respectively, under high nitrogen concentration compared to low nitrogen concentration.

In October, under the NC treatment, changes in moisture levels did not cause significant differences in the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings, and the ¹⁵N abundance was negligible (see Table 1). Under the NL treatment, compared to the control, the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings significantly increased or decreased with changes in moisture levels. The same trend was observed under the NH treatment. Under the moisture control treatment, the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings increased significantly by 9.55%, 19.26%, and 20.93%, respectively, under high nitrogen concentration compared to low nitrogen concentration. Under the increased moisture treatment (WI), the ¹⁵N abundance in the roots, stems, and leaves of Mongolian pine seedlings increased significantly by 44.44%, 36.02%, and 47.02%, respectively, under the NH treatment compared to the NL treatment. Under the decreased moisture treatment (WD), the opposite trend was observed, with the ¹⁵N abundance in the roots, stems, and leaves under the high nitrogen concentration treatment being slightly lower than that under the low nitrogen concentration, with significant differences in the roots and stems.

3.1.2. Changes in Soil ¹⁵N Abundance

As shown in Figure 1a, in August, under the NL treatment, there were no significant differences in the soil ¹⁵N abundance of Mongolian pine seedlings on sandy land across different moisture treatments. However, under the NH treatment, there were significant differences in soil ¹⁵N abundance among the three moisture levels. For the WC, WI, and WD moisture treatments, soil ¹⁵N abundance in Mongolian pine seedlings increased with higher nitrogen concentrations, showing significant differences.

In October (as shown in Figure 1b), under the NC treatment, there were no significant differences in soil ¹⁵N abundance across the three moisture levels. Under the low nitrogen treatment (NL), increased moisture did not significantly affect soil ¹⁵N abundance, but the reduced moisture treatment significantly increased soil ¹⁵N abundance by 22.33%. Under the NH treatment, soil ¹⁵N abundance in Mongolian pine seedlings significantly decreased by 45.11% and 13.62% under the increased and reduced moisture treatments, respectively, compared to the control. For the WC, WI, and WD moisture treatments, soil ¹⁵N abundance in Mongolian pine seedlings increased with higher nitrogen concentrations, showing significant differences.

3.2. Correlation and Variance Analysis of ¹⁵N Abundance in Different Organs and Soil of Mongolian Pine Seedlings Under Various Treatments of Nitrogen Deposition and Precipitation Patterns

3.2.1. Correlation Analysis

As shown in Figure 2, in August, the correlation coefficients between root ¹⁵N abundance and stem ¹⁵N abundance, leaf ¹⁵N abundance and stem ¹⁵N abundance, and leaf ¹⁵N abundance and root ¹⁵N abundance in Mongolian pine seedlings on sandy land were 0.99, 0.99, and 0.97, respectively. This indicated a significant positive correlation among the ¹⁵N abundances in different organs of the seedlings. The correlation coefficient between soil ¹⁵N abundance and leaf ¹⁵N abundance was the highest, followed by those between soil ¹⁵N abundance and stem ¹⁵N abundance and between soil ¹⁵N abundance and root ¹⁵N abundance, all showing significant positive correlations.

As shown in Figure 3, in October, there was a highly significant positive correlation among the ¹⁵N abundances in the roots, stems, and leaves of the Mongolian pine seedlings. Although there were significant positive correlations between the ¹⁵N abundances in the roots, stems, leaves, and soil, the correlation coefficients were lower. The correlation between stem and leaf ¹⁵N abundance and soil ¹⁵N abundance was stronger than that between root ¹⁵N abundance and soil ¹⁵N abundance.

3.2.2. Analysis of Variance

As shown in

Table 2. Analysis of variance (P) for the main effects of month, moisture, nitrogen application, and their interactions on the ¹⁵N abundance of Mongolian pine seedlings and soil.

	M	W	N	M × W	M × N	W × N	M × W × N
Root	**	**	**	**	**	**	**
Stem	**	**	**	**	**	**	**
Leaf	**	**	**	**	**	**	**
Soil	**	**	**	**	**	**	**

**: significant correlation at the p < 0.01 level.

, based on the analysis of variance of month, moisture, and nitrogen application on the ¹⁵N abundance of Mongolian pine seedlings and soil, it was observed that month, moisture, and nitrogen application were the main effects, and their interactions had highly significant (p < 0.01) effects on the ¹⁵N abundance of each organ of Mongolian pine seedlings and soil.

3.3. Effects of Nitrogen Deposition and Changes in Precipitation Patterns on ¹⁵N Uptake by Mongolian Pine

3.3.1. N_dff (%) in Different Organs of Mongolian Pine Seedlings and Soil

As shown in Figure 4a, in August, the N_dff (%) in the roots of Mongolian pine seedlings was generally higher under the increased moisture treatment (WI) compared to other treatments. In the NL treatment, the WI treatment significantly increased the N_dff (%) in the roots by 51.86% and 90.51% compared to the WC and WD treatments, respectively. Under the NH treatment, the increased moisture significantly raised the N_dff (%) in the roots by 114.01% and 43.43% under the WC and WD treatments, respectively. With the same moisture treatment, the NH nitrogen application significantly increased the N_dff (%) in the roots compared to the NL nitrogen application under both the WI and WD treatments. In October, the N_dff (%) in the roots generally showed an increase or decrease corresponding to the changes in moisture levels compared to the control treatment (WC) (Figure 4b). Significant differences in N_dff (%) were observed under different nitrogen application rates in the WC and WI treatments, but not under the WD treatment. In all nitrogen treatments, the N_dff (%) in the roots was significantly higher under the increased moisture treatment (WI) compared to the other two treatments (WC and WD).

In the stems of Mongolian pine seedlings, the N_dff (%) in August was higher in all nitrogen treatments compared to the control treatment (WC). In October, the N_dff (%) in the stems showed a trend of increase or decrease corresponding to the changes in moisture levels, with significant differences observed under different nitrogen treatments (Figure 4c,d). Under the WC moisture treatment, there were no significant differences in N_dff (%) between the NL and NH nitrogen treatments. In October, the effect of the changes in moisture levels on N_dff (%) in the stems was less pronounced under the NL treatment compared to the NH treatment. Under the WD treatment, different nitrogen levels did not result in significant differences in N_dff (%) in the stems of the seedlings.

As shown in Figure 4e,f, the overall trends in N_dff (%) in the leaves of Mongolian pine seedlings in August and October were like those observed in the stems. In August, the N_dff (%) in the leaves was higher under all nitrogen treatments compared to the control treatment (WC). In October, the N_dff (%) in the leaves showed significant differences corresponding to changes in moisture levels under different nitrogen treatments. In August, the variations in N_dff (%) in the leaves were smaller under the NL treatment compared to the NH treatment. Under the WC, WI, and WD treatments, the N_dff (%) in the leaves was significantly higher by 12.56%, 95.35%, and 122.96%, respectively, under the NH treatment compared to the NL treatment. In October, under the WC moisture treatment, the N_dff (%) in the leaves was significantly higher by 22.91% under the NH treatment compared to the NL treatment. Under the WI moisture treatment, the N_dff (%) in the leaves was significantly higher by 50.21% under the high nitrogen treatment compared to the low nitrogen treatment. Under the WD treatment, there were no significant differences in N_dff (%) in the leaves between different nitrogen levels.

The patterns of N_dff (%) changes in the soil were slightly different from those in the organs of Mongolian pine seedlings (Figure 4g,h). Under the low nitrogen treatment (NL), changes in moisture levels did not significantly affect the N_dff (%) in the soil in August. In October, the reduced moisture treatment (ND) slightly increased the N_dff (%) in the soil. In August, the NH treatment significantly increased the N_dff (%) in the soil by 10.34% and 61.57% under the WI and WD treatments, respectively. Significant differences were observed between the two nitrogen treatments under the same moisture treatment. In October, the N_dff (%) in the soil was higher under the NH treatment compared to the NL treatment across all moisture treatments, with significant differences between the WC and WD treatments. Under the NL treatment, the reduced moisture treatment (WD) significantly increased the N_dff (%) in the soil compared to the control treatment (WC), while no significant differences were observed under the increased moisture treatment. Under the NH treatment, changes in moisture levels significantly reduced the N_dff (%) in the soil compared to the control group (NC).

3.3.2. Nitrogen Content and ¹⁵N Absorption

Since the nitrogen content of organs in Mongolian pine seedlings is one of the factors for calculating ¹⁵N absorption, only the results are presented here, focusing on the analysis of ¹⁵N absorption in different organs of the seedlings.

As shown in

Table 3. Nitrogen content and ¹⁵N absorption in different organs of Mongolian pine seedlings under various treatments of nitrogen deposition and precipitation patterns.

Month	Treatments	Nitrogen Content in Organs (mg/plant)			15N Absorption in Organs (mg/plant)
		Root	Stem	Leaf	Root	Stem	Leaf
August	WCNL	3.33 ± 0.76 A a	3.93 ± 0.59 B a	16.99 ± 4.48 A a	0.86 ± 0.20 B a	1.50 ± 0.23 B a	5.35 ± 1.41 A a
	WINL	2.98 ± 0.53 A a	5.23 ± 1.23 A b	19.45 ± 4.15 A a	1.17 ± 0.21 A a	2.18 ± 0.51 A b	6.45 ± 1.38 A b
	WDNL	2.38 ± 1.26 A a	2.92 ± 1.14 B a	10.37 ± 3.28 B b	0.49 ± 0.26 C b	0.77 ± 0.30 C b	2.23 ± 0.70 B b
	WCNH	2.73 ± 1.10 A a	3.91 ± 1.25 B a	13.84 ± 2.88 B a	0.67 ± 0.27 B a	1.46 ± 0.47 B a	4.91 ± 1.02 C a
	WINH	2.76 ± 0.60 A a	7.18 ± 1.26 A a	21.86 ± 4.44 A a	1.46 ± 0.32 A a	4.55 ± 0.79 A a	14.17 ± 2.88 A a
	WDNH	2.74 ± 1.12 A a	3.34 ± 0.70 B a	20.08 ± 5.91 A a	1.01 ± 0.41 B a	1.61 ± 0.34 B a	9.61 ± 2.83 B a
October	WCNL	5.35 ± 1.89 A a	7.40 ± 3.01 A a	30.39 ± 4.14 A a	2.38 ± 0.84 AB a	2.73 ± 1.11 A a	11.2 ± 1.52 A a
	WINL	5.18 ± 3.56 A a	5.14 ± 2.31 AB a	25.97 ± 12.87 A a	2.72 ± 1.87 A a	2.74 ± 1.23 A b	13.73 ± 6.80 A a
	WDNL	2.51 ± 1.36 A a	4.34 ± 0.70 B a	13.19 ± 4.04 B a	0.86 ± 0.47 B a	1.56 ± 0.25 A a	4.32 ± 1.32 B a
	WCNH	2.14 ± 1.15 B b	5.57 ± 0.46 A a	14.43 ± 3.86 B b	1.05 ± 0.56 B b	3.11 ± 0.26 B a	7.13 ± 1.91 B b
	WINH	8.83 ± 6.63 A a	7.72 ± 3.11 A a	27.65 ± 9.65 A a	6.84 ± 5.13 A a	5.70 ± 2.30 A a	21.96 ± 7.66 A a
	WDNH	4.00 ± 3.02 AB a	5.19 ± 1.70 A a	17.38 ± 7.45 B a	1.32 ± 1.00 B a	1.81 ± 0.59 B a	5.66 ± 2.42 B a

Different uppercase letters in the same column indicate significant differences (p < 0.05) between different moisture treatments under the same nitrogen treatment. Different lowercase letters in the same column indicate significant differences (p < 0.05) between different nitrogen treatments under the same moisture treatment.

, in August, under the NL treatment, compared to the control group, changes in moisture levels increased or decreased the ¹⁵N absorption in the roots of Mongolian pine seedlings. Similar trends were observed in the stems and leaves under the NL treatment. Under the NH treatment, compared to the control group, changes in moisture levels consistently resulted in higher ¹⁵N absorption in the roots, stems, and leaves of the seedlings. Under the moisture control treatment (WC), significant differences in ¹⁵N absorption were observed in the roots, stems, and leaves of the seedlings with different nitrogen application levels. In terms of ¹⁵N absorption in the stems and leaves, under the increased moisture treatment (WI), the NH nitrogen treatment significantly increased the ¹⁵N absorption by 108.72% and 119.69%, respectively, compared to the NL nitrogen treatment. Under the reduced moisture treatment (WD), the NH nitrogen treatment significantly increased the ¹⁵N absorption in the roots, stems, and leaves by 106.12%, 109.10%, and 330.94%, respectively, compared to the NL nitrogen treatment.

In October, under the low nitrogen treatment (NL), compared to the moisture control treatment (WC), changes in moisture levels resulted in increased or decreased ¹⁵N absorption in the roots, stems, and leaves of Mongolian pine seedlings. Under the NH treatment, the ¹⁵N absorption in the roots, stems, and leaves increased by 551.43%, 83.28%, and 207.99%, respectively, under the WI treatment compared to the WC treatment, with significant differences observed in the roots and leaves. Under the WI and WD treatments, the high nitrogen treatment consistently increased the ¹⁵N absorption in the roots, stems, and leaves, except for the stems under the WI treatment, where no significant difference was observed. Under the moisture control treatment (WC), the ¹⁵N absorption in the roots and leaves was significantly lower under the high nitrogen treatment compared to the low nitrogen treatment, decreasing by 55.88% and 36.34%, respectively.

3.4. Effects of Nitrogen Deposition and Precipitation Patterns on the ¹⁵N Distribution Ratio in Organs of Mongolian Pine

As shown in Figure 5, the distribution ratios of ¹⁵N in different organs of Mongolian pine seedlings under various nitrogen and moisture treatments in August and October were consistently in the order of leaves > stems > roots. In August, under the NL treatment (Figure 5a), changes in moisture availability (whether an increase or a decrease) affected ¹⁵N distribution. With increased moisture availability, ¹⁵N tended to transfer downward from the leaves to the stems and roots, with a higher retention in the stems compared to the control group and the WD treatment. Conversely, with decreased moisture availability, ¹⁵N in the leaves tended to transfer more towards the roots compared to the control group and the WI treatment. Under the NH treatment in August, compared to the control group (WC), the WD treatment resulted in more ¹⁵N being allocated to the leaves and less to the stems compared to both the control group (WC) and the WI treatment. When moisture was reduced, more ¹⁵N was allocated to the roots of the seedlings compared to increased moisture conditions, although it was still less than in the control group. Overall, under high nitrogen concentration, increased moisture led to relatively more ¹⁵N being allocated to the stems and leaves of the seedlings, while reduced moisture resulted in slightly more ¹⁵N being allocated to the roots.

In October, under the NL treatment (Figure 5c), the amount of ¹⁵N allocated to the roots of the seedlings increased across all moisture treatments compared to August. When moisture was increased, more ¹⁵N was allocated to the leaves compared to the control. With decreased moisture availability, the seedlings tended to allocate more ¹⁵N to the stems and roots compared to the WI treatment and the control group. As shown in Figure 5d, under the NH treatment, more ¹⁵N was allocated to the roots under both the increased and decreased moisture treatments compared to the control group. Compared to the increased and decreased moisture treatments, the control treatment had significantly more ¹⁵N allocated to the stems.

4. Discussion

This study found that atmospheric nitrogen deposition and changes in precipitation patterns influence nitrogen absorption and distribution in Mongolian pine seedlings. Correlation and variance analyses revealed significant correlations in ¹⁵N abundance among different organs of Mongolian pine seedlings, consistent with findings by Yang et al. [53] and Temme et al. [54] in other plants. Moisture and nitrogen addition, along with their interactions, significantly affected the ¹⁵N abundance in different organs of the seedlings. Similar findings were reported by Li et al. [55] in two tropical tree species, which further supported our hypothesis and provided a basis for subsequent discussions.

Increased moisture availability enhanced the ¹⁵N abundance and absorption in the roots, stems, and leaves of the seedlings. This could be due to improved nitrogen absorption efficiency in the roots and other organs with increased moisture availability [56], leading to more nitrogen being transferred to underground organs. In addition, the increased water treatment can also be viewed as years of increased mean annual precipitation (MAP), leading to a decrease in soil bulk density (BD), resulting in increased nitrogen uptake by the root system [57]. Under the low nitrogen treatment, reduced moisture availability can represent years with fewer MAP, resulting in opposite outcomes in ¹⁵N abundance and absorption. Elevated soil BD is also one of the causes we should take into consideration [58], since moisture limitation might restrict root growth and slow nutrient transport to other organs [59]. Similar findings were reported by Bethea et al. [60] in Creeping bentgrass (Agrostis stolonifera L.). Reduced moisture availability decreased nitrogen absorption efficiency, limiting nitrogen utilization, as shown in studies on wheat by Ichir et al. [61], rice by Li et al. [62], and no-till rice by Yang et al. [63] under different soil moisture levels. Climate change may lead to an increase in mean annual temperature (MAT), and the effect of MAT on MAP results in complex precipitation patterns [64]. Our findings further confirmed that moisture is a key factor influencing ¹⁵N dynamics in plants, consistent with Liu et al. [65]. Different nitrogen treatments led to varied responses in ¹⁵N absorption among different organs of the seedlings. Overall, higher nitrogen concentration resulted in greater ¹⁵N absorption in all organs compared to lower nitrogen concentration, likely due to stimulated growth and increased nitrogen demands [66]. Another reason could be that nitrogen deposition as an exogenous addition altered the nutrient supply strategy of the seedlings [67]. However, under moisture-controlled conditions, the increase in ¹⁵N absorption with the high nitrogen treatment compared to the low nitrogen treatment was not significant in the stems in August, further indicating that the interaction between moisture and nitrogen is crucial for ¹⁵N absorption in Mongolian pine seedlings. This understanding helps elucidate the response mechanism of nitrogen absorption in different organs of the seedlings under atmospheric nitrogen deposition and changing precipitation patterns.

Using a ¹⁵N-labeled urea solution to simulate atmospheric nitrogen deposition resulted in changes in ¹⁵N distribution within the plants, generally showing a pattern of leaves > stems > roots, consistent with existing research [68,69]. Under low nitrogen conditions, reduced moisture availability led to more nitrogen being allocated to the roots, possibly due to enhanced nutrient capture by the roots during moisture stress [70], limiting the transfer of nitrogen to above-ground parts [71] and reducing the adverse effects of moisture deficiency [72]. Under high nitrogen conditions, combined with reduced moisture availability, nitrogen allocation to the roots and stems decreased, possibly because nitrogen was not the preferred element under adverse conditions [73]. It may also be due to high nitrogen additions lowering soil pH and creating acidic soils [74], which affects root uptake of nutrients [75]. The allocation of nutrients to the leaves is part of the plant’s survival strategy [76]. Under low nitrogen conditions, changes in moisture availability led to reduced nitrogen allocation to the leaves, making overall nitrogen distribution more dependent on moisture availability. However, under high nitrogen conditions combined with reduced moisture availability, more ¹⁵N accumulated in the leaves, consistent with findings in poplars by Luo et al. [77]. The distribution of nitrogen among different organs in response to environmental changes might relate to the physiological characteristics, tissue structure, and nitrogen demands of the plant [5]. The consistently higher nitrogen allocation to the leaves compared to the roots and stems could be because leaves, being more active organs [78], tend to accumulate more limiting elements [6].

This study also had some limitations due to the pot experiment’s inability to account for seasonal changes in precipitation. To address this, we plan to conduct in situ experiments in the future to deepen our understanding in this field. Additionally, this experiment used one-year-old Mongolian pine seedlings. In future studies, we will include Mongolian pines of various ages, allowing us to comprehensively explore the specific differences in nitrogen allocation mechanisms among pines of different ages under varying atmospheric nitrogen deposition and precipitation patterns. In addition, we plan to incorporate other ecological factors in future experiments, such as CO₂ or O₃ stress, as well as the combined effects of thermal stress, both hot and cold. This approach aims to elucidate more accurately the responses and feedback mechanisms of forest ecosystems to global climate change.

5. Conclusions

This study elucidates the relationship between nitrogen deposition, moisture levels, and nitrogen allocation in Mongolian pine seedlings. The findings reveal a hierarchical nitrogen allocation pattern, with leaves receiving the highest proportion of ¹⁵N, followed by stems and roots, consistent across various treatments. Increased moisture significantly enhances ¹⁵N absorption in all organs, particularly the leaves. This allocation strategy ensures a balanced distribution among the organs, supporting the overall growth and development of the seedlings. Under reduced moisture and low nitrogen conditions, seedlings allocate more nitrogen to the roots and stems, which is crucial for mitigating water deficiency. Conversely, high nitrogen and reduced moisture conditions lead to more nitrogen being allocated to the leaves, highlighting their role in nutrient capture. This adaptive response underscores the resilience of Mongolian pine in the face of environmental challenges. The implications of these findings are profound for ecological restoration, wind erosion control, and agricultural and forestry management, especially in semi-arid regions where climate change poses significant threats. This study advocates for moderate nitrogen deposition combined with increased moisture as a strategy to bolster nitrogen uptake in Mongolian pine seedlings, thereby enhancing their survival and growth in semi-arid environments.